The overall goal for Project I is to elucidate the molecular basis for the targeting of PKA. To achieve this has required a multidisciplinary approach that includes elucidation of the structural motifs that contribute to targeting, identifying interacting partners and targeting motifs associated with D-AKAP1 and2, and generating fluorescent probes in collaboration with Tsien that can query and control PKA signaling in cells and peptide disrupters that can query the importance of specific PKA isoforms. Over the past granting period, in collaboration with Jennings and Core A), we solved NMR structures of the Rla Dimerization/Docking Domain free and bound to a D-AKAP2 peptide and a crystal structure of the Rlla DID Domain bound to the same peptide. Our two major structural goals for the next granting period are defined in Specific Aim I. First is to crystallize the Rla DID domain bound to a set of AKAP peptides and to the A Kinase Binding Domain of D -AKAP1. Here we will also elucidate the importance of the interchain disulfide bond. Our second structural goal, aimed at building higher levels of complexity, is to crystallize the D -AKAP2 AKB Domain bound to the DID domain of Rlla and to the PDZ domains of PDZ-K1 and/or NHERF. Mapping peptide binding sites for interacting proteins using peptide arrays is also an integral part of Specific Aim I. Specific Aim II focuses on mechanisms for targeting AKAPs and PKA-related proteins to the mitochondria and will use Cores B, C, and D. Here we will focus on three proteins: (1) D-AKAP1 which targets to the outer mitochondrial membrane by an N-terminal helical motif, (2) ChChd3 which is a newly discovered PKA substrate that is targeted to the inner membrane by its ChChd domain, and (3) A Kinase interacting Protein 1, AKIP1, which is a newly discovered binding partner for the PKA C-subunit and contributes to its localization in the nucleus. AKIP1 also binds to the Apoptosis Inducing Factor (AIF) which shuttles from the mitochondria to the nucleus in the late stages of apoptosis. In Specific Aim III we continue our efforts to design novel fluorescent reporters for PKA signaling and isoform-specific peptifde disrupters. Our firs goal is to engineer A Kinase Activity Reporters (AKARs) that are targeted to the mitochondrial inner membrane space (IMS). A Kinase Anchoring Disrupter Peptides (AKADs) and PKI inhibitor peptides will also be targeted to the IMS. Our second goal, in a collaboration with Tsien, is to design Reversibly Aggregating GFPs (RAgg-GFPs) that are fused to PKA subunits and AKAPs. These probes can selectively target proteins and protein complexes to inactive fiber-like aggregates that can be rapidly reversed upon photobleachin.